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Heterologous expression of human cytochrome P450 (CYP) 2C19 in Escherichia coli and establishment of RP-HPLC method to serve as activity marker

Pan Y, Mak JW, Ong CE

Biomed Chromatogr, 2013 Jul;27(7):859-65.
PMID: 23386533 DOI: 10.1002/bmc.2872

In this study, a simple and reliable reverse-phase high-performance liquid chromatography (RP-HPLC) method was established and validated to analyze S-mephenytoin 4-hydroxylase activity of a recombinant CYP2C19 system. This system was obtained by co-expressing CYP2C19 and NADPH-CYP oxidoreductase (OxR) proteins in Escherichia coli (E. coli) cells. In addition to RP-HPLC, the expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. The RP-HPLC assay showed good linearity (r(2) = 1.00) with 4-hydroxymephenytoin concentration from 0.100 to 50.0 μm and the limit of detection was 5.00 × 10(-2) μm. Intraday and interday precisions determined were from 1.90 to 8.19% and from 2.20 to 14.9%, respectively. Recovery and accuracy of the assay were from 83.5 to 85.8% and from 95.0 to 105%. Enzyme kinetic parameters (Km , Vmax and Ki ) were comparable to reported values. The presence of CYP2C19 in bacterial membranes was confirmed by immunoblotting and the characteristic absorbance peak at 450 nm was determined in the reduced CO difference spectral assay. Moreover, the activity level of co-expressed OxR was found to be comparable to that of the literature. As a conclusion, the procedures described here have generated catalytically active CYP2C19 and the RP-HPLC assay developed is able to serve as CYP2C19 activity marker for pharmacokinetic drug interaction study in vitro.
Fulltext Involvement of cytochromes p450 in drug-drug interactions: an overview

Ong CE, Teh LK, Ismail R

Med J Malaysia, 2002 Jun;57(2):251-60.
PMID: 24326665

Drug interactions can cause iatrogenic disease. If concurrent medications are taken, the potential exists for a drug interaction to occur. Renewed interest in the topic interactions has been generated by the fatal interactions involving non-sedating histamine H-1 antagonists and the recent intriduction of two therapeutic agents, the selective serotonin reuptake inhibitors (SSRIs) and HIV protease inhibitors, for the treatment of depression and AIDS, respectively. These three therapeutic agents have been implicated in clinically significant drug interactions. The consequences of these interactions vary in clinical significance, extent, and effect. Some interactions are theoretical whereas others may lead to severe iatrogenic adverse experiences including lethal consequences.The purpose of this review is to alert the medical practioner to potential drug interactions that may occur when these drugs are prescribed to patients. The pharmacological basis and clinical signficance of these interactions are reviewed. The pharmacological mechanisms underlying these interactions are illustrative of those that may be involved for many other medications. Doctors should be aware of the potential pitfall that may occur when certain groups of drugs are prescribed with concurrent medications.
Unveiling the Role of Cytochrome P450 (2E1) in Human Brain Specifically in Parkinson's Disease - Literature Review

Shah A, Ong CE, Pan Y

Curr Drug Metab, 2021;22(9):698-708.
PMID: 34325630 DOI: 10.2174/1389200222666210729115151

BACKGROUND: In recent years, the significance of cytochrome P450 enzymes (CYPs) has expanded beyond their role in the liver. Factors such as genetics, environmental toxins, drug biotransformation and underlying diseases mediate the expression of these enzymes. Among the CYP enzymes, CYP2E1, a well-recognized monooxygenase enzyme involved in the metabolism of various endogenous and exogenous substances, plays a crucial role in the brain concerning the development of Parkinson's disease. The expression of CYP2E1 varies in different brain regions making certain regions more vulnerable than others. CYP2E1 expression is inducible which generates tissuedamaging radicals leading to oxidative stress, mitochondrial dysfunction and ultimately neurodegeneration.
OBJECTIVE: Less is understood about the role of CYP2E1 in the central nervous system, therefore the purpose of the study was to investigate the relationship between the expression and activity of CYP2E1 enzyme relevant to Parkinson's disease and to identify whether an increase in the expression of CYP2E1 is associated with neurodegeneration.
METHODS: The objectives of the study were achieved by implicating an unsystematic integrative literature review approach in which the literature was qualitatively analysed, critically evaluated and a new theory with an overall view of the mechanism was presented.
RESULTS: The contribution of CYP2E1 in the development of Parkinson's disease was found to be significant as the negative effects of CYP2E1 overshadowed its protective detoxifying role.
CONCLUSION: Overexpression of CYP2E1 seems detrimental to dopaminergic neurons, therefore, to overcome this, a synthetic biochemical is required, which paves the way for further research and development of valuable biomolecules.
In vitro approaches to investigate cytochrome P450 activities: update on current status and their applicability

Ong CE, Pan Y, Mak JW, Ismail R

Expert Opin Drug Metab Toxicol, 2013 Sep;9(9):1097-113.
PMID: 23682848 DOI: 10.1517/17425255.2013.800482

Cytochromes P450 (CYPs) play a central role in the Phase I metabolism of drugs and other xenobiotics. It is estimated that CYPs can metabolize up to two-thirds of drugs present in humans. Over the past two decades, there have been numerous advances in in vitro methodologies to characterize drug metabolism and interaction involving CYPs.
In vitro modulatory effects of flavonoids on human cytochrome P450 2C8 (CYP2C8)

Pang CY, Mak JW, Ismail R, Ong CE

Naunyn Schmiedebergs Arch Pharmacol, 2012 May;385(5):495-502.
PMID: 22307090 DOI: 10.1007/s00210-012-0731-5

The inhibitory effects of five flavonoids with distinct chemical classes (flavones [luteolin], flavonols [quercetin and quercitrin], and flavanones [hesperetin and hespiridin]) on cDNA-expressed CYP2C8 were investigated. CYP2C8 was co-expressed with NADPH-cytochrome P450 reductase in Escherichia coli and used to characterise potency and mechanism of these flavonoids on the isoform. Tolbutamide 4-methylhydroxylase, a high-performance liquid chromatography-based assay, was selected as marker activity for CYP2C8. Our results indicated that the flavonoids inhibited CYP2C8 with different potency. The order of inhibitory activities was quercetin > luteolin > hesperetin > hesperidin > quercitrin. All of these compounds however exhibited mechanism-based inhibition. A number of structural factors were found to be important for inhibition; these include the molecular shape (volume to surface ratio), the number of hydroxyl groups as well as glycosylation of the hydroxyl group. Quercetin was the most potent inhibitor among the flavonoids examined in this study, and our data suggest that it should be examined for potential pharmacokinetic drug interactions pertaining to CYP2C8 substrates in vivo.
In vitro modulation of naturally occurring flavonoids on cytochrome P450 2A6 (CYP2A6) activity

Tiong KH, Yiap BC, Tan EL, Ismail R, Ong CE

Xenobiotica, 2010 Jul;40(7):458-66.
PMID: 20402563 DOI: 10.3109/00498251003786749

1. The effect of flavonoids on coumarin 7-hydroxylation, an activity marker of an important human liver cytochrome P450 isoform, cytochrome P450 2A6 (CYP2A6), was investigated in this study. 2. Coumarin 7-hydroxylase activity was measured fluorometrically in reaction mixtures containing cDNA-expressed CYP2A6, nicotinamide adenine dinucleotide phosphate generating system and 10 uM coumarin, at various concentrations of flavonoids. 3. Among the 23 compounds tested, most of the active members were from flavonol group of hydroxylated flavonoids, with myricetin being the most potent inhibitor followed by quercetin, galangin, and kaempferol. 4. Further exploration of the inhibition mechanism of these compounds revealed that myricetin, galangin, and kaempferol exhibited mixed-type of inhibition pattern while quercetin was observed to exhibit competitive mode of inhibition. 5. Structure-function analyses revealed that degree of inhibition was closely related to the number and location of hydroxyl groups, glycosylation of the free hydroxyl groups, degree of saturation of the flavane nucleus as well as the presence of the alkoxylated function.
Genetic polymorphism of CYP2C8 in three Malaysian ethnics: CYP2C8*2 and CYP2C8*3 are found in Malaysian Indians

Muthiah YD, Lee WL, Teh LK, Ong CE, Ismail R

J Clin Pharm Ther, 2005 Oct;30(5):487-90.
PMID: 16164496

CYP2C8 is genetically polymorphic. Four variants, CYP2C8*2, CYP2C8*3, CYP2C8*4 and CYP2C8*5, which contain mutations in the coding regions have been reported to exhibit different enzyme activity as compared with CYP2C8*1.
Cytochrome P450 genotype-guided drug therapies: An update on current states

Dong AN, Tan BH, Pan Y, Ong CE

Clin Exp Pharmacol Physiol, 2018 10;45(10):991-1001.
PMID: 29858511 DOI: 10.1111/1440-1681.12978

Over the past 2 decades, knowledge of the role and clinical value of pharmacogenetic markers has expanded so that individualized pre-emptive therapy based on genetic background of patients could be within reach for clinical implementation. This is evidenced from the frequent updating of drug labels that incorporates pharmacogenetic information (where compelling data become available) by the regulatory agencies (such as the US FDA), and the periodical publication of guidelines of specific therapeutic recommendations based on the results of pharmacogenetic tests by the pharmacogenetics working groups or consortiums of professional bodies. Clinical relevance of the cytochrome P450 (CYP) polymorphism related to dose, effectiveness and/or toxicity of key drugs are presented in this review, including that of warfarin, clopidogrel, tricyclic antidepressants, and proton pump inhibitors. Prospect for routine clinical application of CYP genotyping before prescribing drugs is still currently unclear due to challenges and barriers associated with availability of well-defined and validated pharmacogenetic studies, the interpretation, result reporting and potential error of genotype testing, involvement of non-genetic factors, and other patient's demographic and disease conditions. Further studies to provide additional supporting clinical data and acceleration of pharmacogenetic testing standards and techniques should help improve the evidence base needed for clinical utility and hence move the implementation of genotype-guided therapy in clinical practice a step closer to reality.
Inhibition of Human Cytochrome P450 2c8-catalyzed Amodiaquine N-desethylation: Effect of Five Traditionally and Commonly Used Herbs

Muthiah YD, Ong CE, Sulaiman SA, Ismail R

Pharmacognosy Res, 2016 Oct-Dec;8(4):292-297.
PMID: 27695271

In Southeast Asia and many parts of the world, herbal products are increasingly used in parallel with modern medicine.
The current understanding of the interactions between nanoparticles and cytochrome P450 enzymes - a literature-based review

Pan Y, Ong CE, Pung YF, Chieng JY

Xenobiotica, 2019 Jul;49(7):863-876.
PMID: 30028220 DOI: 10.1080/00498254.2018.1503360

Nanoparticles (NPs) have wide spectrum applications in the areas of industry and biomedicine. However, concerns about their toxic and negative impacts on the environments as well as human health have been raised. Cytochrome P450s (CYPs) are involved in endogenous and exogenous metabolism. Modulations of CYP can adversely damage drug metabolism, detoxification of xenobiotics and animal physiology functions. This article focused on NPs-CYP interactions for humans and animals available in the literature. It was found that different NPs process specific inhibitory potencies against CYPs involved in drug metabolism. Moreover, NPs were able to modify the expression of CYPs genes or protein in humans and other animals, which highlighted their detoxification functions. Nonetheless, changes of CYPs responsible for hormone synthesis and metabolism resulted in endocrine disturbances. Hence, there is a need to screen newly developed NPs to evaluate their interactions with CYPs. The future studies should further strategize the in vitro approaches to reveal the molecular mechanisms behind interactions by taking full considerations of the interference of co-factors, buffers, substrates and metabolites with NPs. Moreover, in vivo studies should compare the influences of NPs via different administration routes and different duration of treatments to reveal the physiological significance.
Cytochrome P450 4B1 (CYP4B1) as a target in cancer treatment

Lim S, Alshagga M, Ong CE, Chieng JY, Pan Y

Hum Exp Toxicol, 2020 Jun;39(6):785-796.
PMID: 32054340 DOI: 10.1177/0960327120905959

Cytochrome P450 4B1 (CYP4B1) plays crucial roles in biotransforming of xenobiotics. Its predominant extrahepatic expression has been associated with certain tissue-specific toxicities. However, the expressions of CYP4B1 in various cancers and hence their potential roles in cancer development were inclusive. In this work, existing knowledge on expression and regulation of CYP4B1 gene and protein, catalysis of CYP4B1, association of CYP4B1 with cancers, contradicting findings about human CYP4B1 activities as well as the employing CYP4B1 in suicide gene approach for cancer treatment were reviewed. To date, it appears that there is a wide spectrum of tissue distribution of CYP4B1 with lungs as the predominant sites. Several nuclear receptors are possibly responsible for regulating its gene expression. The involvement of CYP4B1 in cancer was considered via activation of procarcinogens and neovascularization. However, human CYP4B1 was found to be inactive due to a substitution of proline with serine at position 427. Suicide gene approach combining reengineered CYP4B1 and prodrug 4-ipomeanol (4-IPO) has shown a promising potential for targeted cancer therapy. Further studies should focus on the verification of human CYP4B1 catalytic activities. More compounds with similar structure as 4-IPO should be tested to identify more alternative agents for the suicide gene approach in cancer treatment.
Fulltext The CYP2R1 Enzyme: Structure, Function, Enzymatic Properties and Genetic Polymorphism

Dong AN, Tan BH, Pan Y, Ong CE

J Pharm Pharm Sci, 2021;24:94-112.
PMID: 33626316 DOI: 10.18433/jpps31305

Since the discovery of its role in vitamin D metabolism, significant progress has been made in the understanding of gene organisation, protein structure, catalytic function, and genetic polymorphism of cytochrome P450 2R1 (CYP2R1). Located on chromosome 11p15.2, CYP2R1 possesses five exons, unlike most other CYP isoforms that carry nine exons. CYP2R1 crystal structure displays a fold pattern typical of a CYP protein, with 12 a-helices as its structural core, and b-sheets mostly arranged on one side, and the heme buried in the interior part of the protein. Overall, CYP2R1 structure adopts a closed conformation with the B' helix serving as a gate covering the substrate access channel, with the substrate vitamin D3 occupying a position with the side chain pointing toward the heme group. In liver, CYP2R1 25-hydroxylates vitamin D and serves as an important determinant of 25(OH)D level in the tissue and in circulation. While substrate profile has been well studied, inhibitor specificity for CYP2R1 requires further investigation. Both exonic and non-exonic single nucleotide polymorphisms (SNPs) have been reported in CYP2R1, including the CYP2R1*2 carrying Leu99Pro exchange, and a number of non-exonic SNPs with variable functional consequences in gene regulation. A non-exonic SNP, rs10741657, has its causal relationship with diseases established, including that of rickets, ovarian cancer, and multiple sclerosis. The role of other CYP2R1 SNPs in vitamin D deficiency and their causal link to other traits however remain uncertain currently and more studies are warranted to help identify possible physiological mechanisms underlying those complex traits.
Fulltext In vitro and in silico Approaches to Study Cytochrome P450-Mediated Interactions

Tan BH, Pan Y, Dong AN, Ong CE

J Pharm Pharm Sci, 2017;20(1):319-328.
PMID: 29145931 DOI: 10.18433/J3434R

In vitro and in silico models of drug metabolism are utilized regularly in the drug research and development as tools for assessing pharmacokinetic variability and drug-drug interaction risk. The use of in vitro and in silico predictive approaches offers advantages including guiding rational design of clinical drug-drug interaction studies, minimization of human risk in the clinical trials, as well as cost and time savings due to lesser attrition during compound development process. This article gives a review of some of the current in vitro and in silico methods used to characterize cytochrome P450(CYP)-mediated drug metabolism for estimating pharmacokinetic variability and the magnitude of drug-drug interactions. Examples demonstrating the predictive applicability of specific in vitro and in silico approaches are described. Commonly encountered confounding factors and sources of bias and error in these approaches are presented. With the advent of technological advancement in high throughput screening and computer power, the in vitro and in silico methods are becoming more efficient and reliable and will continue to contribute to the process of drug discovery, development and ultimately safer and more effective pharmacotherapy. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.
In vitro effect of important herbal active constituents on human cytochrome P450 1A2 (CYP1A2) activity

Pan Y, Tiong KH, Abd-Rashid BA, Ismail Z, Ismail R, Mak JW, et al.

Phytomedicine, 2014 Oct 15;21(12):1645-50.
PMID: 25442272 DOI: 10.1016/j.phymed.2014.08.003

This study was designed to investigate eight herbal active constituents (andrographolide, asiaticoside, asiatic acid, madecassic acid, eupatorin, sinensetin, caffeic acid, and rosmarinic acid) on their potential inhibitory effects on human cytochrome P450 1A2 (CYP1A2) activity. A fluorescence-based enzyme assay was performed by co-incubating human cDNA-expressed CYP1A2 with its selective probe substrate, 3-cyano-7-ethoxycoumarin (CEC), in the absence or presence of various concentrations of herbal active constituents. The metabolite (cyano-hydroxycoumarin) formed was subsequently measured in order to obtain IC50 values. The results indicated that only eupatorin and sinensetin moderately inhibited CYP1A2 with IC50 values of 50.8 and 40.2 μM, while the other active compounds did not significantly affect CYP1A2 activity with IC50 values more than 100 μM. Ki values further determined for eupatorin and sinensetin were 46.4 and 35.2 μM, respectively. Our data indicated that most of the investigated herbal constituents have negligible CYP1A2 inhibitory effect. In vivo studies however may be warranted to ascertain the inhibitory effect of eupatorin and sinensetin on CYP1A2 activity in clinical situations.
Fulltext Inhibitory potency of 8-methoxypsoralen on cytochrome P450 2A6 (CYP2A6) allelic variants CYP2A6 15, CYP2A6 16, CYP2A6 21 and CYP2A6 22: differential susceptibility due to different sequence locations of the mutations

Tiong KH, Mohammed Yunus NA, Yiap BC, Tan EL, Ismail R, Ong CE

PLoS One, 2014;9(1):e86230.
PMID: 24475091 DOI: 10.1371/journal.pone.0086230

Human cytochrome P450 2A6 (CYP2A6) is a highly polymorphic isoform of CYP2A subfamily. Our previous kinetic study on four CYP2A6 allelic variants (CYP2A6 15, CYP2A6 16, CYP2A6 21 and CYP2A6 22) have unveiled the functional significance of sequence mutations in these variants on coumarin 7-hydroxylation activity. In the present study, we further explored the ability of a typical CYP2A6 inhibitor, 8-methoxypsoralen (8-MOP), in inhibition of these alleles and we hypothesized that translational mutations in these variants are likely to give impact on 8-MOP inhibitory potency. The CYP2A6 variant and the wild type proteins were subjected to 8-MOP inhibition to yield IC50 values. In general, a similar trend of change in the IC50 and Km values was noted among the four mutants towards coumarin oxidation. With the exception of CYP2A6 16, differences in IC50 values were highly significant which implied compromised interaction of the mutants with 8-MOP. Molecular models of CYP2A6 were subsequently constructed and ligand-docking experiments were performed to rationalize experimental data. Our docking study has shown that mutations have induced enlargement of the active site volume in all mutants with the exception of CYP2A6 16. Furthermore, loss of hydrogen bond between 8-MOP and active site residue Asn297 was evidenced in all mutants. Our data indicate that the structural changes elicited by the sequence mutations could affect 8-MOP binding to yield differential enzymatic activities in the mutant CYP2A6 proteins.
Effect of eurycomanone on cytochrome P450 isoforms CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 in vitro

Pan Y, Tiong KH, Abd-Rashid BA, Ismail Z, Ismail R, Mak JW, et al.

J Nat Med, 2014 Apr;68(2):402-6.
PMID: 23881640 DOI: 10.1007/s11418-013-0794-8

Eurycomanone, an active constituent isolated from Eurycoma longifolia Jack, was examined for modulatory effects on cytochrome P450 (CYP) isoforms CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2E1 and CYP3A4 using in vitro assays. The IC50 value was determined to assess the potencies of modulation for each CYP isoform. Our results indicated that eurycomanone did not potently inhibit any of the CYP isoforms investigated, with IC50 values greater than 250 μg/ml. Hence there appears to be little likelihood of drug-herb interaction between eurycomanone or herbal products with high content of this compound and CYP drug substrates via CYP inhibition.
Inhibitory effects of cytochrome P450 enzymes CYP2C8, CYP2C9, CYP2C19 and CYP3A4 by Labisia pumila extracts

Pan Y, Tiong KH, Abd-Rashid BA, Ismail Z, Ismail R, Mak JW, et al.

J Ethnopharmacol, 2012 Sep 28;143(2):586-91.
PMID: 22885070 DOI: 10.1016/j.jep.2012.07.024

Labisa pumila (LP), popularly known with its local name, Kacip Fatimah, is a well known herb grown in Indochina and Southeast Asia and is traditionally used to regain energy after giving birth in women. The propensity of LP to cause drug-herb interaction via cytochrome P450 (CYP) enzyme system has not been investigated.
Fulltext Functional characterization of cytochrome P450 2A6 allelic variants CYP2A6*15, CYP2A6*16, CYP2A6*21, and CYP2A6*22

Tiong KH, Yiap BC, Tan EL, Ismail R, Ong CE

Drug Metab. Dispos., 2010 May;38(5):745-51.
PMID: 20139165 DOI: 10.1124/dmd.109.031054

Variation in CYP2A6 levels and activity can be attributed to genetic polymorphism and, thus, functional characterization of allelic variants is necessary to define the importance of CYP2A6 polymorphism in humans. The aim of the present study was to investigate the reported alleles CYP2A6*15, CYP2A6*16, CYP2A6*21, and CYP2A6*22, in terms of the functional consequences of their mutations on the enzyme catalytic activity. With use of the wild-type CYP2A6 cDNA as template, site-directed mutagenesis was performed to introduce nucleotide changes encoding K194E substitution in CYP2A6*15, R203S substitution in CYP2A6*16, K476R substitution in CYP2A6*21, and concurrent D158E and L160I substitutions in CYP2A6*22. Upon sequence verification, the CYP2A6 wild-type and mutant constructs were individually coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. A kinetic study using a coumarin 7-hydroxylase assay indicated that CYP2A6*15 exhibited higher V(max) than the wild type, whereas all mutant constructs, except for variant CYP2A6*16, exhibited higher K(m) values. Analysis of the V(max)/K(m) ratio revealed that all mutants demonstrated 0.85- to 1.05-fold differences from the wild type, with the exception of variant CYP2A6*22, which only portrayed 39% of the wild-type intrinsic clearance. These data suggested that individuals carrying the CYP2A6*22 allele are likely to have lower metabolism of CYP2A6 substrate than individuals expressing CYP2A6*15, CYP2A6*16, CYP2A6*21, and the wild type.
Functional role of Ile264 in CYP2C8: mutations affect haem incorporation and catalytic activity

Singh R, Ting JG, Pan Y, Teh LK, Ismail R, Ong CE

Drug Metab. Pharmacokinet., 2008;23(3):165-74.
PMID: 18574320

The work described in this study aimed to express CYP2C8 wild-type and mutant proteins in bacterial expression system and to use the expressed proteins to investigate the structural and functional consequences of a reported allele CYP2C8(*)4 (carrying Ile264Met substitution) on protein activity. Ile264 was replaced by three different amino acids resulting in three mutant constructs, 2C8I264M, 2C8I264R and 2C8I264D. The presence of isoleucine at position 264 in CYP2C8 was found to be important for proper haem insertion and protein folding; whereas bulkier or charged residues were highly disruptive resulting in inactive proteins with minimum spectral and catalytic activities. This was evidenced from the low levels of Soret peak at 450 nm and negligible levels of tolbutamide methylhydroxylase activity. Kinetic study using paclitaxel indicated that all three mutants exhibited only 9.7 to 35.4% of the activity level observed in the wild-type. In addition, the mutants were more sensitive to proteinase K digestion, indicating a possible alteration of conformation. The combined effects of protein instability and compromised catalytic activity resulted in defective CYP2C8 protein which may have clinical implications in carriers of CYP2C8*4, particularly in terms of their capacity to clear potent drugs and their susceptibility to adverse drug reactions.
Heterologous expression of human cytochromes P450 2D6 and CYP3A4 in Escherichia coli and their functional characterization

Pan Y, Abd-Rashid BA, Ismail Z, Ismail R, Mak JW, Ong CE

Protein J, 2011 Dec;30(8):581-91.
PMID: 22001938 DOI: 10.1007/s10930-011-9365-6

This study aimed to express two major drug-metabolizing human hepatic cytochromes P450 (CYPs), CYP2D6 and CYP3A4, together with NADPH-cytochrome P450 oxidoreductase (OxR) in Escherichia coli and to evaluate their catalytic activities. Full length cDNA clones of both isoforms in which the N-terminus was modified to incorporate bovine CYP17α sequence were inserted into a pCWori(+) vector. The modified CYP cDNAs were subsequently expressed individually, each together with OxR by means of separate, compatible plasmids with different antibiotic selection markers. The expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. Enzyme activities were examined using high performance liquid chromatography (HPLC) assays with probe substrates dextromethorphan and testosterone for CYP2D6 and CYP3A4, respectively. Results from immunoblotting demonstrated the presence of both CYP proteins in bacterial membranes and reduced CO difference spectra of the cell preparations exhibited the characteristic absorbance peak at 450 nm. Co-expressed OxR also demonstrated an activity level comparable to literature values. Kinetic parameters, K(m) and V(max) values determined from the HPLC assays also agreed well with literature values. As a conclusion, the procedures described in this study provide a relatively convenient and reliable means of producing catalytically active CYP isoforms suitable for drug metabolism and interaction studies.

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